Characterization of mammary tumor cell lines from wild type and vitamin D3 receptor knockout mice

1,25-Dihydroxyvitamin D(3) (1,25D(3)), the active metabolite of vitamin D(3), inhibits breast cancer cell growth in vivo and in vitro. To examine mechanisms of 1,25D(3) induced growth arrest and apoptosis, cell lines were established from DMBA induced mammary tumors derived from vitamin D(3) receptor knockout (VDRKO) and wild type (WT) mice. Two VDRKO (KO240, KO288) and two WT (WT145, WT276) cell lines were selected and characterized. All four cell lines express cytokeratins indicative of an epithelial origin, as well as vimentin, which is expressed in many transformed cell lines. The tumorigenicity of the cells was confirmed in vivo as all four cell lines form estrogen responsive tumors in nude mice. Both WT cell lines express the VDR protein and are sensitive to growth inhibition by 1,25D(3) at doses as low as 1 nM. Flow cytometric analysis indicated that 1,25D(3) induces G(0)/G(1) arrest and apoptosis in the WT cell lines. In contrast, both cell lines established from tumors that developed in VDRKO mice lack VDR mRNA and protein. Cells from WT mice exhibit 1,25D(3) inducible transcriptional activity, as measured by reporter gene assays, but cells from VDRKO mice do not. Cells from VDRKO mice are also completely resistant to 1,25D(3) mediated growth arrest and apoptosis over the range of 0.01-100 nM 1,25D(3). VDRKO cells are also resistant to the synthetic vitamin D(3) analogs EB1089 and CB1093 that are more potent growth inhibitors than 1,25D(3) in WT cells. This data conclusively demonstrate that the induction of cell cycle arrest and apoptosis in breast cancer cells by 1,25D(3), EB1089 and CB1093 is dependent on the nuclear VDR. Cells lacking VDR remain sensitive to growth arrest mediated by 9-cis retinoic acid, a ligand for the retinoid x receptor which can heterodimerize with the VDR. Sensitivity to apoptosis induced by the DNA damaging agent etoposide is not altered in VDRKO cells, indicating that VDR ablation does not impair apoptotic pathways in general. All four cell lines display equal sensitivity to tamoxifen induced growth arrest. These estrogen responsive, transformed cell lines which differentially express the VDR provide a novel model system for identification of the mechanisms by which 1,25D(3) regulates proliferation and apoptosis in breast cancer cells.